A cell cryopreservation composition and uses thereof

Abstract

The present application provides a kind of cell cryopreservation composition and its application.The cell cryopreservation composition contains IL-2, basic cryopreservation fluid and any one or more selected from IL-7, IL-15 and IFN-γ.The immune cells cryopreserved using the composition can achieve higher instant and overnight survival rate after recovery, and the killing effect on target cells is more significant.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to a cell cryopreservation composition and its application. Background Technology

[0002] Immunotherapy has received increasing attention in recent years. Adoptive immunotherapy has become an important means of tumor immunotherapy. Currently, the immune cells used clinically include CAR-T, TCR-T, TIL, DC-CIK, NK, CAR-NK, LAK, DNT (double-negative T cells), CTL, and CAR-M (macrophages). Among them, several CAR-T cell products have been launched in overseas markets. In the Chinese market, there are also CAR-T cell products and Akiserenone injection targeting CD19, which are used to treat relapsed or refractory indolent non-Hodgkin lymphoma after receiving two or more lines of systemic therapy.

[0003] Currently, the primary route of administration for immunocellular therapy is intravenous delivery. After preparation, immunocellular drugs are typically resuspended at a specific density in a cell cryopreservation composition, such as a cell cryopreservation solution, to form a cryopreservation formulation. This formulation is then cryopreserved in liquid nitrogen, transported to the patient's ward, thawed, and then intravenously reinfused into the patient. In this process, the cell viability and activity after cryopreservation and thawing are crucial for the subsequent therapeutic effect, determining whether a sufficient number of cells remain in the body to effectively proliferate and kill tumors after reinfusion. To date, all commercially available immunocellular drug products worldwide are CAR-T cells, derived from the patient's autologous peripheral blood PBMCs. For example, Novartis's US-marketed CAR-T product, Kymriah, contains components in its cryopreservation formulation including berberine electrolyte A, dextran, sodium chloride, dextran, human serum albumin, and dimethyl sulfoxide. In addition, there are also relatively mature commercial cryopreservation solutions, such as BioLifeSolutions' CryoStor series of cryopreservation solutions, and CryoStor cryopreservation solutions have been used in Juno's CAR-T cell drug Breyanzi, which is marketed in the United States.

[0004] On the one hand, these cryopreservation solutions still have a significant impact on CAR-T cells, and the survival rate of cells thawed after cryopreservation still needs further improvement. On the other hand, for T cells not derived from peripheral blood and immune cells other than T cells, the cryopreservation effect of existing cell cryopreservation solutions needs to be further improved, and other cryopreservation solutions that are more suitable for different sources and types of immune cells need to be developed. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a cell cryopreservation composition and its uses. The inventors have discovered that compositions formed by the cell cryopreservation solution CryoStor and various different cytokines exhibit superior cryopreservation effects. Therefore, the objective of this invention is to provide a cell cryopreservation composition with superior cryopreservation effects.

[0006] A first aspect of the present invention provides a cell cryopreservation composition comprising IL-2, a basal cryopreservation solution, and one or more selected from IL-7, IL-15, and IFN-γ, wherein the basal cryopreservation solution is selected from... and Any one of them.

[0007] In one or more embodiments, the cell cryopreservation composition is an immune cell cryopreservation composition.

[0008] In one or more embodiments, the cell cryopreservation composition contains any one of the following: (1) IL-7, (2) IL-7 and IFN-γ, (3) IL-15 and IFN-γ, (4) IL-7, IL-15 and IFN-γ.

[0009] In one or more embodiments, the IL-2 concentration is 10-600 IU / mL, preferably 20-500 IU / mL, 100-500 IU / mL, or 200-500 IU / mL.

[0010] In one or more embodiments, the concentration of IL-7 is 0-600 IU / mL, preferably 20-500 IU / mL, 20-100 IU / mL, or 20-50 IU / mL.

[0011] In one or more embodiments, the concentration of IL-15 is 0-600 IU / mL, preferably 20-500 IU / mL, 20-300 IU / mL, or 20-200 IU / mL.

[0012] In one or more embodiments, the concentration of IFN-γ is 0-300 IU / mL, preferably 10-200 IU / mL, 10-150 IU / mL, or 10-100 IU / mL.

[0013] In one or more embodiments, the base cryopreservation solution is

[0014] In one or more embodiments, the cell cryopreservation composition further comprises one or more of carbohydrates, serum albumin, glycine, vitamin E, and electrolytes, wherein the carbohydrates are monosaccharides selected from glucose, fructose, and dextrose, and / or disaccharides selected from lactose and trehalose, preferably dextrose and / or trehalose.

[0015] In one or more embodiments, the cell cryopreservation composition contains any of the following: (1) carbohydrates, glycine and vitamin E, (2) serum albumin, glycine and electrolytes, (3) carbohydrates, serum albumin and vitamin E, (4) carbohydrates, serum albumin, glycine, vitamin E and electrolytes.

[0016] In one or more embodiments, the concentration of monosaccharides (e.g., dextrose) in the cell cryopreservation composition is 0-500 mmol / L, preferably 10-200 mmol / L, and more preferably 50-100 mmol / L.

[0017] In one or more embodiments, the concentration of disaccharides (e.g., trehalose) in the cell cryopreservation composition is 0-2.5 mol / L, preferably 0.5-2.0 mol / L, more preferably 1.0-2.0 mol / L.

[0018] In one or more embodiments, the serum albumin is human serum albumin.

[0019] In one or more embodiments, the concentration of serum albumin in the cell cryopreservation composition is 0-150 μg / mL, preferably 20-100 μg / mL, for example 20-60 μg / mL or 60-100 μg / mL.

[0020] In one or more embodiments, the concentration of glycine in the cell cryopreservation composition is 0-70 μg / mL, preferably 5-50 μg / mL, more preferably 5-25 μg / mL.

[0021] In one or more embodiments, the concentration of vitamin E in the cell cryopreservation composition is 0-12.5 μg / mL, preferably 1-10 μg / mL, more preferably 3-10 μg / mL.

[0022] In one or more embodiments, the electrolyte comprises one or more of the following: sodium chloride, sodium gluconate, sodium acetate, potassium chloride, and magnesium chloride. In one or more embodiments, the concentration of sodium chloride in the electrolyte is 0-200 mol / mL, preferably 50-150 mol / mL; the concentration of sodium gluconate is 0-50 mol / mL, preferably 10-30 mol / mL; the concentration of sodium acetate is 0-50 mol / mL, preferably 10-30 mol / mL; the concentration of potassium chloride is 0-20 mol / mL, preferably 1-10 mol / mL; and the concentration of magnesium chloride is 0-10 mol / mL, preferably 1-5 mol / mL. Preferably, the electrolyte is a compound electrolyte injection solution of PulseA.

[0023] In one or more embodiments, the electrolyte content in the cell cryopreservation composition is 0-35 v / v%, preferably 20-30 v / v.

[0024] In a preferred embodiment, the cell cryopreservation composition contains CryoStor10 and ingredients selected from any of the following groups:

[0025] (a) IL-2 at 15-25 IU / mL, IL-7 at 450-550 IU / mL, and IFN-γ at 45-55 IU / mL.

[0026] (b) 15-25 IU / mL IL-2, 450-550 IU / mL IL-15, 150-250 IU / mL IFN-γ, 1.5-2.5 mol / L trehalose, 45-55 μg / mL glycine, and 0.5-1.5 μg / mL vitamin E.

[0027] (c) 450-550 IU / mL IL-2, 15-25 IU / mL IL-7, 15-25 IU / mL IL-15, 5-15 IU / mL IFN-γ, 80-120 μg / mL human serum albumin, 20-30 μg / mL glycine, and 15-25 v / v% electrolytes.

[0028] (d) 150-250 IU / mL IL-2, 80-120 IU / mL IL-7, 0.2-0.8 mol / L trehalose, 50-70 μg / mL human serum albumin, and 2.0-4.5 μg / mL vitamin E.

[0029] (e) 50-150 IU / mL IL-2, 45-55 IU / mL IL-7, 150-250 IU / mL IL-15, 80-120 IU / mL IFN-γ, 0.5-1.5 mol / L trehalose, 15-25 μg / mL human serum albumin, 2-8 μg / mL glycine, 8.0-12.0 μg / mL vitamin E, and 25-35 v / v% electrolytes.

[0030] A second aspect of the present invention provides a cell cryopreservation formulation comprising the cell cryopreservation composition and cells described in any of the embodiments herein.

[0031] In one or more embodiments, the concentration of cells in the formulation is 5 × 10⁻⁶. 6 From 1×10⁻⁶ cells / mL to 1×10⁻⁶ cells / mL 8 Cells / mL, preferably 3×10 7 Up to 1×10 8 Cells / mL, more preferably about 5 × 10⁻⁶ 7per mL.

[0032] In one or more embodiments, the cell is an immune cell.

[0033] In one or more embodiments, the immune cells include any one or more selected from CAR-T, TCR-T, TIL, DC-CIK, NK, CAR-NK, LAK, DNT, CTL and CAR-M, preferably CAR-T cells and / or TIL cells.

[0034] A third aspect of the present invention provides a method for cryopreserving cells using a cell cryopreservation composition described in any embodiment herein, comprising the following steps:

[0035] (1) Mix the cells with the cell cryopreservation composition described in any embodiment of this document, and

[0036] (2) Gradually cool the mixture to -20 to -150°C, and

[0037] Optionally (3) the mixture is stored in liquid nitrogen.

[0038] In one or more embodiments, the concentration of cells in the cell cryopreservation composition is 5 × 10⁻⁶. 6 From 1×10⁻⁶ cells / mL to 1×10⁻⁶ cells / mL 8 Cells / mL, preferably about 5 × 10⁻⁶ 7 per mL.

[0039] In one or more embodiments, step (2) includes: gradually cooling the mixture to -50 to -120°C, preferably -70 to -100°C, more preferably about -90°C.

[0040] In one or more embodiments, the cell is an immune cell.

[0041] In one or more embodiments, the immune cells include any one or more selected from CAR-T, TCR-T, TIL, DC-CIK, NK, CAR-NK, LAK, DNT, CTL and CAR-M, preferably CAR-T cells and / or TIL cells.

[0042] A fourth aspect of the present invention provides the use of the cell cryopreservation composition described in any embodiment herein in the preparation of a cell-containing reagent, the cell cryopreservation composition being used for cryopreserving cells.

[0043] In one or more embodiments, the cell-containing reagent is a cell cryopreservation formulation comprising cells and the cell cryopreservation composition, wherein preparing the formulation includes the step of cryopreserving cells using the cell cryopreservation composition.

[0044] In one or more embodiments, the cell-containing reagent is a reagent containing revived cells, wherein preparing the reagent includes the steps of cryopreserving cells using a cell cryopreservation composition and the step of cell revival.

[0045] In one or more embodiments, the cryopreserved cells include the steps of:

[0046] (1) Mix the cells with the cell cryopreservation composition described in any of the embodiments herein.

[0047] (2) Gradually cool the mixture to -20 to -150°C, and

[0048] Optionally (3) the mixture is stored in liquid nitrogen.

[0049] In one or more embodiments, the mixing ratio of cells to the cell cryopreservation composition is 5 × 10⁻⁶. 6 From 1×10⁻⁶ cells / mL to 1×10⁻⁶ cells / mL 8 Cells / mL, preferably about 5 × 10⁻⁶ 7 per mL.

[0050] In one or more embodiments, step (2) includes: gradually cooling the mixture to -50 to -120°C, preferably -70 to -100°C, more preferably about -90°C.

[0051] In one or more embodiments, the cell resuscitation step includes: (1) thawing the frozen cells in a 37°C water bath, preferably for 1-2 minutes, and optionally (2) culturing the cells in a culture medium for at least 2 hours, preferably overnight. The culture medium is a cell culture medium containing 0-800 IU / mL IL-2, 0-10 v / v% serum, and a basal medium (e.g., X-VIVO 15).

[0052] In one or more embodiments, the cell is an immune cell.

[0053] In one or more embodiments, the immune cells include any one or more selected from CAR-T, TCR-T, TIL, DC-CIK, NK, CAR-NK, LAK, DNT, CTL and CAR-M, preferably CAR-T cells and / or TIL cells.

[0054] Advantages of this invention: The cell cryopreservation composition provided by this invention significantly improves the immediate and overnight survival rates of cells after thawing compared to existing cell cryopreservation solutions. Furthermore, the cytotoxicity of immune effector cells thawed using the cell cryopreservation composition of this invention against target cells is also significantly enhanced compared to existing cell cryopreservation solutions. Attached Figure Description

[0055] Figure 1 The RTCA killing curve of CAR-T cells frozen in cryopreservation composition 5-9 against target cells HGC-27 after thawing. Detailed Implementation

[0056] To improve the survival rate of cryopreserved cells after thawing, the inventors developed a cell cryopreservation composition comprising at least IL-2, a basal cryopreservation solution, and one or more selected from IL-7, IL-15, and IFN-γ. This composition allows for the cryopreservation of cells (e.g., immune cells) at higher concentrations, and cells thawed using this composition achieve higher immediate viability, with significantly higher viability even after overnight thawing and culture. Immune cells cryopreserved using the cryopreservation composition of this invention exhibit a more significant killing effect on target cells after thawing.

[0057] In this invention, the term "IL-2" refers to interleukin-2, a cytokine with multicellular origin (mainly produced by activated T cells) and multipotent functions (primarily promoting lymphocyte growth, proliferation, and differentiation). It plays a crucial role in the body's immune response and antiviral infection, stimulating the proliferation of T cells initiated by specific antigens or mitogenic factors; activating T cells and promoting cytokine production; stimulating NK cell proliferation, enhancing NK cell killing activity and producing cytokines, inducing LAK cell production; promoting B cell proliferation and antibody secretion; and activating macrophages. In the cell cryopreservation composition of this invention, the IL-2 concentration is 10, 20, 30, 50, 100, 200, 500, or 600 IU / mL, or any two of the above values. For example, the IL-2 concentration in the cell cryopreservation composition is 20-500 IU / mL, 100-500 IU / mL, or 200-500 IU / mL.

[0058] The terms "IL-7" and "IL-15" refer to interleukin-7 and interleukin-15, respectively, which are two cytokines in the chemokine family. In the cell cryopreservation composition of the present invention, when contained, the concentrations of IL-7 and IL-15 are each independently 10, 20, 30, 50, 100, 200, 500, or 600 IU / mL, or any range between two of the above values. For example, the concentration of IL-7 in the cell cryopreservation composition is 20-500 IU / mL, 20-100 IU / mL, or 20-50 IU / mL. For example, the concentration of IL-15 in the cell cryopreservation composition is 20-500 IU / mL, 20-300 IU / mL, or 20-200 IU / mL.

[0059] The term "IFN-γ" refers to interferon-gamma, which has antiviral, antitumor, and immunomodulatory effects. It can regulate the expression levels of 30 genes, producing a variety of cellular responses. In the cell cryopreservation compositions of the present invention, the concentration of IFN-γ is 0, 10, 20, 30, 50, 100, 200, or 300 IU / mL, or any range between two of the above values. For example, the concentration of IFN-γ in the cell cryopreservation compositions is 10-200 IU / mL, 10-150 IU / mL, or 10-100 IU / mL.

[0060] In specific embodiments, the cell cryopreservation composition described herein contains any one of the following: (1) IL-7, (2) IL-7 and IFN-γ, (3) IL-7, IL-15 and IFN-γ.

[0061] In this article, the basic cryopreservation solution includes 2 (CryoStor2) 5 (CryoStor5) 10 (CryoStor10) and CSB (CryoStorCSB) and other commercially available products This series of cryopreservation solutions are pre-prepared cryopreservation solutions manufactured under cGMP without animal-derived components and with a defined composition containing 10%, 5%, or 2% USP-grade dimethyl sulfoxide (DMSO). They are primarily used to mitigate temperature-induced molecular stress responses during cryopreservation and thawing, and are suitable for cells sorted from various tissue types. In this paper, CryoStor 10 is preferably used as the base cryopreservation solution, and its specific volume can be adjusted to the required final volume based on actual conditions. However, this invention does not preclude the use of other series of base cryopreservation solutions or other base cryopreservation solutions within the CryoStor series.

[0062] The cell cryopreservation composition may also contain one or more of carbohydrates, serum albumin, glycine, vitamin E, and electrolytes. In a preferred embodiment, the cell cryopreservation composition further contains any one of the following: (1) carbohydrates, glycine, and vitamin E; (2) serum albumin, glycine, and electrolytes; (3) carbohydrates, serum albumin, and vitamin E; (4) carbohydrates, serum albumin, glycine, vitamin E, and electrolytes.

[0063] The sugars are monosaccharides selected from glucose, fructose, and dextrose, and / or disaccharides selected from lactose and trehalose, preferably dextrose and / or trehalose. In the cell cryopreservation compositions of the present invention, when contained, the concentration of monosaccharides (e.g., dextrose) is 10, 20, 50, 80, 90, 100, 150, 200, 300, 400, or 500 mmol / L, or a range between any two of the above values. For example, the concentration of monosaccharides (e.g., dextrose) in the cell cryopreservation compositions is 10-200 mmol / L or 50-100 mmol / L. In the cell cryopreservation compositions of the present invention, when contained, the concentration of disaccharides (e.g., trehalose) is 0.1, 0.5, 1.0, 1.2, 1.5, 1.8, 2.0, 2.2, or 2.5 mol / L, or a range between any two of the above values. For example, the concentration of disaccharides (e.g., trehalose) in the cell cryopreservation composition is 0.5-2.0 mol / L or 1.0-2.0 mol / L.

[0064] The serum albumin is preferably human serum albumin, but serum albumin from other species, such as bovine serum albumin, is not excluded. In the cell cryopreservation composition of the present invention, when present, the concentration of serum albumin is 10, 20, 50, 60, 80, 100, 120, or 150 μg / mL, or a range between any two of the above values. For example, the concentration of serum albumin in the cell cryopreservation composition is 20-100 μg / mL, 20-60 μg / mL, or 60-100 μg / mL.

[0065] In the cell cryopreservation composition of the present invention, when glycine is present, the concentration is 5, 10, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 μg / mL, or a range between any two of the above values. For example, the concentration of glycine in the cell cryopreservation composition is 5-50 μg / mL or 5-25 μg / mL.

[0066] In the cell cryopreservation composition of the present invention, when present, the concentration of vitamin E is 0.3, 0.5, 1, 1.5, 2, 2.5, 3, 5, 8, 10, 12, 15, or any two of the above values. For example, the concentration of vitamin E in the cell cryopreservation composition is 1-10 μg / mL or 3-10 μg / mL.

[0067] In this document, the electrolyte can be an organic or inorganic metal salt commonly used for cryopreservation or injection, preferably comprising one or more of the following: sodium chloride, sodium gluconate, sodium acetate, potassium chloride, and magnesium chloride. Exemplarily, in the electrolyte, the concentration of sodium chloride is 0-200 mol / mL, preferably 50-150 mol / mL; the concentration of sodium gluconate is 0-50 mol / mL, preferably 10-30 mol / mL; the concentration of sodium acetate is 0-50 mol / mL, preferably 10-30 mol / mL; the concentration of potassium chloride is 0-20 mol / mL, preferably 1-10 mol / mL; and the concentration of magnesium chloride is 0-10 mol / mL, preferably 1-5 mol / mL. Preferably, the electrolyte is a compound electrolyte injection solution containing 90 mol / mL sodium chloride, 23 mol / mL sodium gluconate, 27 mol / mL sodium acetate (C2H3NaO2·3H2O), 5 mol / mL potassium chloride, and 1.5 mol / mL magnesium chloride (MgCl2·6H2O). The electrolyte may also contain sodium hydroxide. In the cell cryopreservation composition of the present invention, when the electrolyte is present, the content is 0-35 v / v%, preferably 20-30 v / v%.

[0068] In a specific embodiment, the cell cryopreservation composition contains 15-25 IU / mL of IL-2, 450-550 IU / mL of IL-7, 45-55 IU / mL of IFN-γ, and CryoStor10. Preferably, it contains 20 IU / mL of IL-2, 500 IU / mL of IL-7, 50 IU / mL of IFN-γ, with the balance being CryoStor10.

[0069] In another specific embodiment, the cell cryopreservation composition contains 15-25 IU / mL IL-2, 450-550 IU / mL IL-15, 150-250 IU / mL IFN-γ, 1.5-2.5 mol / L trehalose, 45-55 μg / mL glycine, 0.5-1.5 μg / mL vitamin E, and CryoStor10. Preferably, it contains 20 IU / mL IL-2, 500 IU / mL IL-15, 200 IU / mL IFN-γ, 2.0 mol / L trehalose, 50 μg / mL glycine, 1 μg / mL vitamin E, and the balance being CryoStor10.

[0070] In another specific embodiment, the cell cryopreservation composition contains 450-550 IU / mL of IL-2, 15-25 IU / mL of IL-7, 15-25 IU / mL of IL-15, 5-15 IU / mL of IFN-γ, 80-120 μg / mL of human serum albumin, 20-30 μg / mL of glycine, 15-25 v / v% of electrolyte, and CryoStor10. Preferably, it contains 500 IU / mL of IL-2, 20 IU / mL of IL-7, 20 IU / mL of IL-15, 10 IU / mL of IFN-γ, 100 μg / mL of human serum albumin, 25 μg / mL of glycine, 20 v / v% of Peptamin A electrolyte, with the balance being CryoStor10.

[0071] In another specific embodiment, the cell cryopreservation composition contains 150-250 IU / mL of IL-2, 80-120 IU / mL of IL-7, 0.2-0.8 mol / L of trehalose, 50-70 μg / mL of human serum albumin, 2.0-4.5 μg / mL of vitamin E, and CryoStor10. Preferably, it contains 200 IU / mL of IL-2, 100 IU / mL of IL-7, 0.5 mol / L of trehalose, 60 μg / mL of human serum albumin, 3 μg / mL of vitamin E, and the balance being CryoStor10.

[0072] In another specific embodiment, the cell cryopreservation composition contains 50-150 IU / mL of IL-2, 45-55 IU / mL of IL-7, 150-250 IU / mL of IL-15, 80-120 IU / mL of IFN-γ, 0.5-1.5 mol / L of trehalose, 15-25 μg / mL of human serum albumin, 2-8 μg / mL of glycine, 8.0-12.0 μg / mL of vitamin E, 25-35 v / v% of electrolytes, and CryoStor10. The preferred formulation contains 100 IU / mL IL-2, 50 IU / mL IL-7, 200 IU / mL IL-15, 100 IU / mL IFN-γ, 1.0 mol / L trehalose, 20 μg / mL human serum albumin, 5 μg / mL glycine, 10 μg / mL vitamin E, 30 v / v% of PulseA compound electrolyte, with the balance being CryoStor10.

[0073] This invention also provides a cell cryopreservation formulation comprising the cell cryopreservation composition and cells described herein. The formulation is a cryopreservation formulation, for example, at -196°C (stored in liquid nitrogen). Cells suitable for the cryopreservation solution and cryopreservation method of this invention are preferably immune cells. In this context, immune cells include any one or more selected from CAR-T, TCR-T, TIL, DC-CIK, NK, CAR-NK, LAK, DNT, CTL, and CAR-M, preferably CAR-T cells (e.g., mesothelin-targeting CAR-T) and / or TIL cells. The aforementioned cells can be corresponding cells prepared by conventional methods in the art, see, for example, CN110746505A and WO2021239083A1. The immune cell cryopreservation composition of this invention can cryopreserve high-density immune cells, for example, 5 × 10⁶ cells / year. 6 From 1×10⁻⁶ cells / mL to 1×10⁻⁶ cells / mL 8 Cells / mL, preferably 3×10 7 Up to 1×10 8 Cells / mL, more preferably about 5 × 10⁻⁶ 7 per mL.

[0074] The cell cryopreservation composition of the present invention can be used for cryopreserving cells and preparing the cell cryopreservation formulations described herein. A method for cryopreserving cells using the cell cryopreservation composition described herein includes the following steps: (1) mixing cells with the cell cryopreservation composition described herein, and (2) gradually cooling the mixture to -20 to -150°C, and optionally (3) storing the mixture in liquid nitrogen. The gradual cooling is any programmed cooling known in the art for freezing cells (e.g., immune cells), such as cooling using a programmed cooling device. An exemplary programmed cooling process includes: cooling to approximately -40°C, heating to approximately -12°C, and cooling to approximately -90°C. Further, the cooling in step 2) includes: 2.1) cooling to approximately -4°C, 2.2) cooling to approximately -40°C, 2.3) heating to approximately -12°C, 2.4) cooling to approximately -40°C, and 2.5) cooling to approximately -90°C. More specifically, the cryopreservation method includes: mixing the cryopreservation composition described herein with cells (e.g., immune cells) and cooling the mixture, the cooling process including: 2.1) cooling at about 1.0°C / min to about -4°C, 2.2) cooling at about 25.0°C / min to about -40°C, 2.3) warming at about 10.0°C / min to about -12°C, 2.4) cooling at about 1.0°C / min to about -40°C, and 2.5) cooling at about 10.0°C / min to about -90°C. Although the examples use immune cells as an example, the immune cell cryopreservation composition of the present invention is not limited to cryopreserving immune cells, but is also applicable to other cells, such as other animal cells.

[0075] This invention also provides a method for preparing cells, including the step of thawing cells from the cell cryopreservation formulation described herein. Those skilled in the art can select appropriate reagents and methods based on the type, state, freezing time, etc., of the cells to be thawed. Exemplarily, cell thawing includes: (1) thawing the cell cryopreservation formulation in a 37°C water bath, preferably for 1-2 minutes, and optionally (2) culturing the cells in a culture medium for at least 2 hours, preferably overnight. The culture medium can be any culture medium known in the art for culturing cells, particularly immune cells (e.g., T cells). Exemplary culture media contain 0-800 IU / mL IL-2, 0-10 v / v% serum, and basal medium (e.g., X-VIVO 15). The method for preparing cells may also include the step of thawing cells as described above to prepare the cell cryopreservation formulation.

[0076] The present invention also provides the use of cell cryopreservation compositions in the preparation of reagents containing cells (e.g., immune cells), the cell cryopreservation compositions being used for cryopreserving cells. In some embodiments, when the cell-containing reagent is a cell cryopreservation formulation, the formulation comprises cells and the cell cryopreservation composition, and the preparation of the formulation includes the steps described herein for cryopreserving cells using the cell cryopreservation composition. In other embodiments, when the cell-containing reagent is a reagent containing revived cells, the preparation of the reagent further includes the cell revival steps described herein.

[0077] The present invention also provides a kit comprising the cell cryopreservation composition described in any embodiment herein. The kit further comprises a cryopreservation container and / or instructions for using the cell cryopreservation composition to cryopreserve cells.

[0078] The present invention will be described below by way of specific embodiments. It should be understood that these embodiments are merely illustrative and are not intended to limit the scope of the invention. The above-described technical features of the present invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form preferred technical solutions. Unless otherwise stated, the methods, reagents and instruments used in the embodiments are conventional methods, reagents and instruments in the art.

[0079] Example

[0080] Example 1: Preparation of cell cryopreservation composition

[0081] Use BioLifeSolutions as shown in Table 1 10 (CS10) Prepare cryopreservation composition.

[0082] Table 1: Formulation of cryopreservation compositions

[0083]

[0084] Cryopreservation composition 6: Commercial CryoStor 10 cryopreservation solution alone;

[0085] Frozen Composition 7: The frozen formulation of Novartis' marketed CAR-T product Kymriah is as follows: 31.25% Pulmatrix A compound electrolyte injection, 31.25% 5% dextran / sodium chloride injection, 10% dextran 40 (dissolved in dextran injection), 20% human serum albumin and 7.5% dimethyl sulfoxide (all by volume / volume percentage);

[0086] Frozen composition 8: supplemented with 600 IU / rhIL-2 of cold storage 10;

[0087] Cryopreservation composition 9: CN110959606B, the cryopreservation solution for immune effector cells as described in claim 1.

[0088] Example 2, Preparation of immune effector cells

[0089] 1. Preparation of CAR-T cells

[0090] The CAR-T cells of the present invention are mesothelin-targeting CAR-T cells, specifically prepared according to the methods described in Examples 1-2 of CN110746505A.

[0091] 2. Preparation of TIL cells

[0092] The TILs of the present invention can be TILs prepared by conventional methods in the art. Specifically, cervical cancer tumor tissue is taken and prepared according to the method described in Example 5 "Preparation of TILs by Reinfusion" in WO2021239083A1.

[0093] Example 3: Survival rate of CAR-T cells after cryopreservation and thawing

[0094] 1. Cryopreservation of CAR-T cells

[0095] Take the CAR-T cells prepared in Example 2 and use cryopreservation compositions 1-8 described in Example 1 at a ratio of 5 × 10⁻⁶. 7 Cells were resuspended at 1-2 mL / mL in cryovials and stored using a ThermoScientific temperature-programmed freezer. TM 7453) After cooling to -90℃, it is placed in liquid nitrogen for cryopreservation. The cooling program settings for the programmed cooling instrument are as follows:

[0096] Step 1: Place the cell sample at 4°C and cool it down to 4°C in the temperature-programmed cooling chamber;

[0097] Step 2: Place the cell sample in a programmed cooling device at a cooling rate of 1.0℃ / minute until it reaches -4℃;

[0098] Step 3: Cooling rate 25.0℃ / min, cooling to -40℃;

[0099] Step 4: Heating rate 10.0℃ / min, heating to -12℃;

[0100] Step 5: Cooling rate 1.0℃ / minute, cooling to -40℃;

[0101] Step 6: Cooling rate 10.0℃ / min, cooling to -90℃;

[0102] Step 7: End.

[0103] 2. CAR-T cell resuscitation and survival rate detection

[0104] Three months after cryopreservation, CAR-T cells cryopreserved with different cryopreservation compositions were placed in a 37°C water bath and vigorously shaken until thawed within 1-2 minutes. The cells were then transferred to 1.5 mL EP tubes, centrifuged at 300 × g for 8 minutes, the supernatant was discarded, and the cell pellet was resuspended in 1 mL PBS. 100 μL of the cell suspension was used to detect the immediate viability using a cell counter. The thawed CAR-T cells were then cultured overnight in basal medium containing 500 IU / mL IL-2, 5 v / v% human AB serum, and X-VIVO 15 to the final volume. Viability was then detected again, and the results are shown in Table 2.

[0105] Table 2: Survival rate of different CAR-T cells after thawing in different cryopreservation compositions

[0106]

[0107]

[0108] The results showed that the immediate survival rate and overnight survival rate of CAR-T cells cryopreserved using cryopreservation compositions 1-5 were significantly higher than those of CAR-T cells cryopreserved using cryopreservation solutions 6-9. Specifically, the immediate survival rate of CAR-T cells cryopreserved using cryopreservation composition 5 was 120.7%, 127.7%, 118.7%, and 126.6% of that of the control cryopreservation compositions 6, 7, 8, and 9, respectively; the overnight survival rate was 129.0%, 132.0%, 125.4%, and 133.6% of that of cryopreservation compositions 6, 7, 8, and 9, respectively.

[0109] Example 4: Survival rate of TIL cells after cryopreservation and thawing

[0110] 1. Cryopreservation of TIL cells

[0111] Take the TIL cells prepared in Example 2 and use cryopreservation compositions 1-8 described in Example 1 at a ratio of 5 × 10⁻⁶. 7 Cells were resuspended at 1-2 mL / mL in cryovials and stored using a Thermo Scientific temperature-programmed freezer. TM 7453) After cooling, place it in liquid nitrogen for freezing. The cooling program of the programmed cooling device is the same as that in Example 3.

[0112] 2. TIL cell resuscitation and survival rate detection

[0113] Three months after cryopreservation, TIL cells cryopreserved with different cryopreservation compositions were placed in a 37°C water bath and vigorously shaken until thawed within 1-2 minutes. The cells were then transferred to 1.5 mL EP tubes, centrifuged at 300 × g for 8 minutes, the supernatant was discarded, and the cell pellet was resuspended in 1 mL PBS. 100 μL of the cell suspension was used to detect the immediate viability using a cell counter. The revived TIL cells were then cultured overnight in basal medium containing 500 IU / mL IL-2, 5 v / v% human AB serum, and X-VIVO 15 to the final volume. Viability was then detected again, and the results are shown in Table 3.

[0114] Table 3: Survival rate of different TIL cells after thawing in different cryopreservation compositions

[0115] 1 92.3 66.2 2 91.5 69.3 3 92.7 71.3 4 93.2 72.4 5 95.6 79.2 6 89.1 56.3 7 83.9 54.7 8 91.2 61.6 9 85.4 57.0

[0116] Table 3 shows that, compared with TIL cells thawed after cryopreservation using cryopreservation compositions 6-9, TIL cells thawed using cryopreservation compositions 1-5 had higher or similar immediate viability after thaw, while their overnight viability after thaw was significantly higher than that of cells thawed using cryopreservation compositions 6-9. Specifically, the immediate viability of CAR-T cells thawed using cryopreservation composition 5 was 107.3%, 114.0%, 104.8%, and 111.9% of the control compositions 6, 7, 8, and 9, respectively; the overnight viability after thaw was 140.7%, 144.8%, 128.6%, and 138.9% of the control compositions 6, 7, 8, and 9, respectively.

[0117] Example 5: Detection of CAR-T cell killing power after resuscitation

[0118] CAR-T cells that were cryopreserved overnight using cryopreservation composition 5-9 were used as target cells in the mesothelin-positive gastric cancer cell line HGC-27, and RTCA killing assays were performed according to the following steps:

[0119] (1) Zeroing: Add 50 μL of DMEM or 1640 culture medium to each well, place it in the instrument, select step 1, and zero it;

[0120] (2) Target cell plating: SKOV-3 cells were plated at 10 per well. 4 50 μL of cells were seeded in a plate containing the detection electrode and left to stand for a few minutes to allow the cells to stabilize before placing them in the instrument to begin step 2, cell culture.

[0121] (3) Add effector cells: After culturing target cells for 20 hours, pause step 2, add effector cells, 50 μL per well, add effector cells at an effector-to-target ratio of 4:1, start step 3, continue co-culturing for more than 50 hours, and observe the cell proliferation curve.

[0122] The results are as follows Figure 1 The results showed that, compared with CAR-T cells cryopreserved and then revived using cryopreservation compositions 6-9, CAR-T cells cryopreserved and then revived using cryopreservation composition 5 had a more significant killing effect on target cells HGC-27 under the condition of an effector-to-target ratio of 4:1.

[0123] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details based on all the teachings disclosed, and all such changes are within the scope of protection of this invention. The full scope of this invention is given by the appended claims and any equivalents thereof.

Claims

1. A cell cryopreservation composition comprising IL-2, a basal cryopreservation solution, and a combination of any one of the following: (1) IL-7, (2) IL-7 and IFN-γ, (3) IL-15 and IFN-γ, (4) IL-7, IL-15, and IFN-γ, wherein the basal cryopreservation solution is selected from CryoStor. ® 2. CryoStor ® 5. CryoStor ® 10 and CryoStor ® Any of the CSBs, wherein the cell cryopreservation composition is an immune cell cryopreservation composition, and the immune cells are T cells or TIL cells.

2. The cell cryopreservation composition of claim 1, wherein the base cryopreservation solution is CryoStor. ® 10.

3. The cell cryopreservation composition according to claim 1, characterized in that, The IL-2 concentration is 10-600 IU / mL, and / or The concentration of IL-7 is 0-600 IU / mL, and / or The concentration of the IL-15 is 0-600 IU / mL, and / or The concentration of IFN-γ is 0-300 IU / mL.

4. The cell cryopreservation composition according to claim 1, characterized in that, The IL-2 concentration is 20-500 IU / mL, 100-500 IU / mL, or 200-500 IU / mL, and / or The concentration of IL-7 is 20-500 IU / mL, 20-100 IU / mL, or 20-50 IU / mL, and / or The concentration of IL-15 is 20-500 IU / mL, 20-300 IU / mL, or 20-200 IU / mL, and / or The concentration of IFN-γ is 10-200 IU / mL, 10-150 IU / mL, or 10-100 IU / mL.

5. The cell cryopreservation composition according to claim 1 or 2, characterized in that, The cell cryopreservation composition also contains one or more of carbohydrates, serum albumin, glycine, vitamin E, and electrolytes, wherein the carbohydrates are monosaccharides selected from glucose, fructose, and dextrose, and / or disaccharides selected from lactose and trehalose.

6. The cell cryopreservation composition according to claim 5, characterized in that, The cell cryopreservation composition further comprises any one of the following: (1) carbohydrates, glycine and vitamin E, (2) serum albumin, glycine and electrolytes, (3) carbohydrates, serum albumin and vitamin E, (4) carbohydrates, serum albumin, glycine, vitamin E and electrolytes.

7. The cell cryopreservation composition according to claim 5, characterized in that, The concentration of the disaccharide is 0-2.5 mol / L, and / or The concentration of the monosaccharide is 0-500 mmol / L, and / or The concentration of serum albumin is 0-150 μg / mL, and / or The concentration of glycine is 0-70 μg / mL, and / or The concentration of vitamin E is 0-12.5 μg / mL, and / or The electrolyte content is 0-35 v / v.

8. The cell cryopreservation composition according to claim 7, characterized in that, The concentration of the disaccharide is 0.5-2.0 mol / L, and / or The concentration of the monosaccharide is 10-200 mmol / L, and / or The concentration of serum albumin is 20-100 μg / mL, and / or The concentration of glycine is 5-50 μg / mL, and / or The concentration of vitamin E is 1-10 μg / mL, and / or The electrolyte content is 20-30 v / v.

9. The cell cryopreservation composition according to claim 8, characterized in that, The concentration of the disaccharide is 1.0-2.0 mol / L, and / or The concentration of the monosaccharide is 50-100 mmol / L, and / or The concentration of serum albumin is 20-60 μg / mL or 60-100 μg / mL, and / or The concentration of glycine is 5-25 μg / mL, and / or The concentration of vitamin E is 3-10 μg / mL.

10. The cell cryopreservation composition according to claim 1 or 2, characterized in that, The cell cryopreservation composition contains CryoStor10 and components selected from any of the following groups: (a) IL-2 at 15-25 IU / mL, IL-7 at 450-550 IU / mL, and IFN-γ at 45-55 IU / mL. (b) 15-25 IU / mL IL-2, 450-550 IU / mL IL-15, 150-250 IU / mL IFN-γ, 1.5-2.5 mol / L trehalose, 45-55 μg / mL glycine, and 0.5-1.5 μg / mL vitamin E. (c) 450-550 IU / mL IL-2, 15-25 IU / mL IL-7, 15-25 IU / mL IL-15, 5-15 IU / mL IFN-γ, 80-120 μg / mL human serum albumin, 20-30 μg / mL glycine, and 15-25 v / v % electrolytes. (d) 150-250 IU / mL IL-2, 80-120 IU / mL IL-7, 0.2-0.8 mol / L trehalose, 50-70 μg / mL human serum albumin, and 2.0-4.5 μg / mL vitamin E. (e) 50-150 IU / mL IL-2, 45-55 IU / mL IL-7, 150-250 IU / mL IL-15, 80-120 IU / mL IFN-γ, 0.5-1.5 mol / L trehalose, 15-25 μg / mL human serum albumin, 2-8 μg / mL glycine, 8.0-12.0 μg / mL vitamin E, and 25-35 v / v % electrolytes.

11. The cell cryopreservation composition according to claim 5, characterized in that, The serum albumin is human serum albumin, and / or The electrolyte comprises one or more of the following: sodium chloride, sodium gluconate, sodium acetate, potassium chloride, and magnesium chloride.

12. A cell cryopreservation formulation comprising the cell cryopreservation composition and cells as described in any one of claims 1-6.

13. The cell cryopreservation formulation according to claim 12, characterized in that, The concentration of cells in the formulation is 5 × 10⁻⁶. 6 From 1×10⁻⁶ cells / mL to 1×10⁻⁶ cells / mL 8 per mL, and / or The immune cells are T cells or TIL cells.

14. A method for cryopreserving cells using the cell cryopreservation composition according to any one of claims 1-11, comprising the following steps: (1) Mixing the cells with the cell cryopreservation composition according to any one of claims 1-11, and (2) Gradually cool the mixture to -20 to -150 °C, and Optionally (3) the mixture is stored in liquid nitrogen.

15. The method as described in claim 14, characterized in that, The cell concentration in the cell cryopreservation composition is 5 × 10⁻⁶. 6 From 1×10⁻⁶ cells / mL to 1×10⁻⁶ cells / mL 8 per mL, and / or Step (2) includes: gradually cooling the mixture to -50 to -120 °C, and / or The cells are immune cells, specifically T cells or TIL cells.

16. Use of the cell cryopreservation composition of any one of claims 1-11 in the preparation of a cell-containing reagent, wherein the cell cryopreservation composition is used to cryopreserve cells, the cells being immune cells, and the immune cells being T cells or TIL cells.

17. The use as described in claim 16, characterized in that, The cell-containing reagent is a cell cryopreservation formulation, which comprises cells and the cell cryopreservation composition, wherein preparing the formulation includes the step of cryopreserving cells using the cell cryopreservation composition, or The cell-containing reagent is a reagent containing revived cells, wherein the preparation of the reagent includes the steps of freezing cells using a cell cryopreservation composition and the step of cell revival.

18. The use as described in claim 16, characterized in that, The cryopreserved cells comprise the steps of: (1) mixing the cells with the cell cryopreservation composition according to any one of claims 1-6; (2) gradually cooling the mixture to -20 to -150 °C; and optionally (3) storing the mixture in liquid nitrogen. The cell revival includes the steps of: (1) thawing the frozen cells in a 37°C water bath, and optionally (2) culturing the cells in a culture medium for at least 2 hours.

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